p53 functions as a central node for organizing whether the cell responds to various types and levels of stress via apoptosis, cell cycle arrest, senescence, DNA repair, cell metabolism, autophagy and aging. While the exact molecular events for p53-mediated choice of cell fate are still insufficiently explained, p53 controlled transactivation of target genes comprises an essential event in each stress response pathway. As a transcription factor, p53 demands an exquisitely complicated network of control and fine-tuning mechanisms to ensure correct, differentiated responses to the various stress signals encountered by cells. p53 was the first non-histone protein known to be regulated by acetylation (Gu and Roeder, 1997). The acetylation levels of p53 are significantly enhanced in response to stress, and correlate well with p53 activation and stabilization in all cell types in respond to almost every type of stress. Following our early findings of C-terminus p53 acetylation, we and others recently showed that p53 is also acetylated by Tip60/hMOF at residue K120 within the DNA binding domain (Tang et al., 2006; Sykes et al., 2006). K120 acetylation is crucial for p53-mediated apoptosis but has no obvious effect on p21 expression, an essential target of p53-mediated growth arrest. More recently, we have identified K164 as a new site for in vivo acetylation of p53 by GBP/p300 and evaluated its function in p21 activation. Although acetylation defects at each individual site (K164, K120, and C-terminus) can be compensated by the modification of other sites, loss of acetylation at all these major sites completely abolishes p53's ability to activate its mediated cell growth arrest and apoptosis. These studies demonstrate an indispensible role of acetylation in p53 regulation (Tang et al., 2008). On the other hand, p53 is tightly regulated by Mdm2 and its related protein Mdmx. Nonetheless, the molecular mechanisms by which p53 activity is controlled by Mdm2 and Mdmx are complex (Brooks and Gu, 2006). Mdm2 and MdmX are structurally related proteins and physiological levels of both proteins are required in a non redundant manner to balance p53 activity during embryonic development. Notably, our recent studies show that acetylation of p53 abrogates Mdm2- and Mdmx-mediated repression by blocking the recruitment of Mdm2 or Mdmx to p53 responsive promoters (Tang et al., 2008). Our study identifies anti-repression as a major mechanism for acetylation-mediated p53 activation in stress responses. In summary, for the past 10 years, our lab has made significant contributions to understand this dynamic pathway of p53 regulation by protein modifications. The central hypothesis to be tested here is that protein modification of p53 such as acetylation plays a major part in the scope of controlling p53 function by directly affecting the interactions of p53 with Mdm2/Mdmx or other cellular regulators in vivo, which enables p53 to activate transcription in a promoter-specific manner. It includes the following two specific aims. In Aim 1 we will elucidate the precise roles of p53 acetylation in releasing the repression of p53 from Mdm2 and Mdmx. In Aim 2, we will identify additional cellular factors involved in anti-repression mechanisms and investigate the roles of these factors in modulating p53 activities in stress responses.